1. Field of the Invention
This invention is directed to a process for isolating, from microbial refractile inclusion bodies, recombinant proteins and polypeptides that are difficult to solubilize and separate by previously known techniques. The process involves disruption of the microbial biomass, collection of refractile bodies, and solubilization of refractile body protein. Solubilization is effected by a combination of physical, chemical, and enzymatic means, with protection of free sulfhydryl groups of the recombinant protein by use of reversible of irreversible blocking agents. The free amino groups of proteins are then derivatized by reaction with cyclic acid anhydrides and individual protein derivatives separated using molecular sieves and ion exchange chromatography. The sulfhydryl and amino groups of the separated proteins are then deblocked.
Included in the invention is the preparation of diagnostic devices comprising solid supports coated with the separated recombinant protein antigens, and the use of the diagnostic devices for assays of antibodies in body fluids.
2. Brief Description of the Background Information
Recombinant DNA technology has permitted the expression of foreign (heterologous) proteins in microbial and other host cells. In many instances, high expression of recombinant proteins leads to the formation of high molecular weight aggregates, often referred to as "inclusion bodies" or "refractile bodies" (Old and Primrose, Principles of Gene Manipulation, Third Edition, Blackwell Scientific Publishers, Oxford, 1985, pp. 289-290). The inclusion bodies fall into two categories: first, paracrystalline arrays in which the protein presumably is in a stable conformation, although not necessarily native; and second, amorphous aggregates that contain partially and completely denatured proteins, as well as aberrant proteins synthesized as a result of inaccurate translation. Such aggregates of heterologous protein constitute a significant portion of the total cell protein.
Although inclusion bodies probably afford protection to proteins against endogenous proteases, they do present problems of extraction and purification, as they are very difficultly soluble in aqueous buffers. In most instances, denaturants and detergents (e.g., guanidine hydrochloride, urea, sodium dodecylsulfate (SDS), Triton X-100) have to be used to extract the protein. For proteins of pharmaceutical interest, particularly for parenteral administration, the use of detergents and denaturants is undesirable because it is difficult to remove them completely from isolated proteins, particularly if the proteins possess extensive hydrophobic domains. Further, if guanidine hydrochloride (guanidine.HCl), a strong denaturant, is employed, renaturation of the isolated proteins may be difficult, if not impossible, and the resultant heterologous proteins may be biologically inactive due to incorrect folding or conformation. In addition, if urea, a relatively weak denaturant, is used as the extractant, modification of some amino acid residues may occur.
Another problem in the recovery of the desired proteins which are in the form of refractile bodies is the need, not only to separate refractile proteins from other host cellular materials but also subsequently to remove refractile body protein contaminants from the desired refractile body heterologous protein. The second problem is probably due to the strong attraction that refractile body proteins have one for another, due perhaps to ionic attractions or hydrophobic bonding.
In a series of closely related patents (Builder, et al., U.S. Pat. Nos. 4,551,502; Olson, et al., 4,511,503; Jones, et al., 4,512,922; and Olson, 4,518,526), the inventors teach a process for converting E. coli refractile body recombinant proteins from the natural or induced insoluble state into soluble forms. The techniques use a combination of membrane disruption methods such as sonication or homogenization under high pressure, coupled to solubilization of proteins and strong denaturants such as guanidine.HCl and in ionic detergents such as SDS. After such treatments the recombinant proteins are refolded by buffer exchange into a relatively weak denaturant such as urea in the presence of a reducing agent such as 2-mercaptoethanol (buffer exchange may be preceded by sulfitolysis of proteins by contacting proteins with sulfite and a mild oxidizing reagent). The recombinant proteins are then isolated using ion exchange and molecular sieve chromatography in the presence of buffered urea. Thus, according to these patents, denaturants must necessarily be present throughout, and subsequent to, the process for recovering refractile body proteins.
Two patents from Kinsella et al. (U.S. Pat. Nos. 4,168,262 and 4,348,479) and two technical reports from the same group (Shetty et al., Biochemical Journal, 191:269-272 (1980); Shetty et al., Journal of Argricultural and Food Chemistry, 30:1166-1172 (1982)) teach a process of separating microbial proteins in bulk from nucleoprotein complexes. The process comprises disruption of the biomass by physical means in the absence of detergents of denaturating reagents. This is followed by centrifugation to remove cell debris, derivatization of the water-soluble proteinaceous material-nucleic acid mixture with an organic dicarboxylic acid anhydride such as citraconic or maleic anhydride, and subjecting the derivatized proteins (freed of insoluble cell debris by centrifugation) to isoelectric precipitation at pH 4.0-4.5. Next, the blocking N-acyl groups are removed by hydrolysis at acid pH, the protein solution is dialyzed to reomve salts, and the nucleic acid- depleted bulk proteins are isolated by lyophilization or isoelectric precipitation. It is important to note that the goal of the two Kinsella et al. patents and the Shetty et al. technical reports is to isolate bulk microbial proteins in a form suitable for human consumption. The purpose of the N-acylation step is to separate the desired bulk proteins from microbial nucleic acid contaminants.
Citraconylation has also been used to solubilize proteins from mammalian cells. Eshhar et al., Europoean Journal of Immunology, 1:323-329 (1971), reported that 60-70% of membrane proteins from rat thymocytes and lymphocytes were solubilized by citraconylation, and that these protein derivatives were antigenically inactive until decitraconylated at pH 5. Only 10-20% of the original antigenicity was recovered by deacylation. Lundahl et al., Biochim. Biophys. Acta, 379: 304-316 (1975), disclosed the use of citraconylation to solubilize a portion of water-insoluble human erythrocyte membrane proteins. Citraconylated proteins were further separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or on hydroxyapatite columns.
Kinsella et al., International Journal of Peptide and Protein Research, 18:18-25 (1981), disclosed that citraconylation of proteins is not limited to N-acylation of the primary amino groups of lysine residues. Citraconic anhydride also alkylates some free sulfhydryl groups, leading to cross-linking, and thereby aggregation, of proteins.
Light, Biotechniques, 3:298-308 (1985) disclosed that the solubility of unfolded proteins increases, and aggregation is avoided, when amino groups are derivatized with citraconic anhydride.
Snyder et al. Carbohydrate Research, 105:87-93 (1982) use citraconylation to solubilize human salivary proteins, and to dissociate mucin glycoproteins from extraneous proteins. No attempt was made to separate indivdual citraconylated proteins.
It would be useful to provide a method that solves the problems in solubilizing and separating microbial recombinant heterologous proteins by using procedures which, in their various aspects, succeed in solubilizing the proteins, removing contaminating host cellular proteins, and separating individual recombinant proteins in forms that are active and appropriate in biological and immunoassays, and that are soluble and biologically active in the absence of any denaturant or detergent.